Reaction Wood Anatomical Traits and Hormonal Profiles in Poplar Bent Stem and Root

Abstract

Reaction wood (RW) formation is an innate physiological response of woody plants to counteract mechanical constraints in nature, reinforce structure and redirect growth toward the vertical direction. Differences and/or similarities between stem and root response to mechanical constraints remain almost unknown especially in relation to phytohormones distribution and RW characteristics. Thus, Populus nigra stem and root subjected to static non-destructive mid-term bending treatment were analyzed. The distribution of tension and compression forces was firstly modeled along the main bent stem and root axis; then, anatomical features, chemical composition, and a complete auxin and cytokinin metabolite profiles of the stretched convex and compressed concave side of three different bent stem and root sectors were analyzed. The results showed that in bent stems RW was produced on the upper stretched convex side whereas in bent roots it was produced on the lower compressed concave side. Anatomical features and chemical analysis showed that bent stem RW was characterized by a low number of vessel, poor lignification, and high carbohydrate, and thus gelatinous layer in fiber cell wall. Conversely, in bent root, RW was characterized by high vessel number and area, without any significant variation in carbohydrate and lignin content. An antagonistic interaction of auxins and different cytokinin forms/conjugates seems to regulate critical aspects of RW formation/development in stem and root to facilitate upward/downward organ bending. The observed differences between the response stem and root to bending highlight how hormonal signaling is highly organ-dependent.

Keywords: UHPLC-MS/MS; auxins; bending stress; cytokinins; metabolite profiling.

Figure 1

Model of the mechanical forces distribution along P. nigra bent stem and root. Mechanical forces distribution along the stem (A) and root (B) main axis, at the beginning (t_i) and the end (t_f) of bending treatment. Average value (MPa) of the mechanical force magnitudes are indicated on the corresponded concave (negative values – compression) and convex (positive values – tension) sides of three bent sectors (ABS, BS, and BBS). ABS, above bending sector; BS, bending sector; BBS, below bending sector.

Figure 2

Anatomical bent stem and root cross-sections. Cross-sections of the convex and concave sides of three bent stem and root sectors (ABS, BS, and BBS) stained with Toluidine Blue O. Scale bar = 20 μm. Magnification shows secondary wood fiber cell wall characteristics. Scale bar = 2 μm.

Figure 3

Carbohydrate and lignin content of P. nigra stem and root. Total carbohydrate and lignin content from CX and CE sides of ABS, BS, and BBS are indicated in the (A) and (B) for the bent stem and in the (C) and (D) for the bent root. Percentage value represents the mean of five independent samples ±SD analyzed by Py-GC/MS. Significant differences (post-hoc LSD-tests, p < 0.05) among the CX sectors are indicated by lower letters whereas those among CE sectors by capital letter. Significant differences (post-hoc LSD-tests, p < 0.05) between sides of the same sector are indicated by asterisk. Differences in total lignin amount are indicated above the histograms while differences in Syringyl- (S-), Guaiacyl- (G-), and p-Hydroxyphenyl- (H-) types lignin are indicated inside the histograms. ABS, above bending sector; BS, bending sector; BBS, below bending sector; CX, convex side; CE, concave side.

Figure 4

Auxin metabolites profiling in different bent sides and sectors of P. nigra stem and root. Concentrations of IAA (A), IAGlu (B), IAAsp (C), and oxIAA (D) were analyzed by UHPLC-MS/MS. The values are expressed in pg. mg⁻¹ of dry weight (DW). Data represent the mean of three independent extractions ±SD. All significant differences (post-hoc LSD-tests, p < 0.05) between the three bent sectors in convex and concave sides are indicated by minuscule and capital letter, respectively. Significant differences (post-hoc LSD-tests, p < 0.05) between sides of the same sector are indicated by asterisk and continuous line for root or dashed line for stem. ABS, above bending sector; BS, bending sector; BBS, below bending sector; CX, convex side; CE, concave side.

Figure 5

Amounts of different CK-types/forms and conjugates in P. nigra bent stem and root. The total amount of tZ-, cZ-, DHZ-, and iP-types is illustrated in the graph for stem (A) and root (B) and the corresponding level of each CK-form/conjugate in the heat maps (C,D). Different colors in the heat map indicate the abundance of each CK-form/conjugate in the different samples (n = 3): green and red colors indicate, respectively, relative high and low abundance while white color indicates value below the limit of detection (<LOD). Significant differences (post-hoc LSD-tests, p < 0.05) between the three bent sectors (ABS, BS, and BBS) of the same sides (CX or CE) are indicated, respectively, by lower and capital letter, while those between sides of the same sector are indicated by asterisk. ABS, above bending sector; BS, bending sector; BBS, below bending sector; CX, convex side; CE, concave side.

Figure 6

Ratio of total/active CKs to IAA. Stem (A) and root (B) ratio between the total content of CKs and IAA are indicated by black squared (total CKs/IAA) while ratio between the content of CKs active forms (sum of CK bases and ribosides) and IAA are indicated by gray diamonds (active CKs/IAA). Error bars indicate SD (n = 3). Significant differences (Student’s t test, p < 0.05) between the three bent sectors (ABS, BS, and BBS) along CX and CE sides are indicated by lower and capital letter, respectively, while those between sides of the same sector are indicated by asterisk. ABS, above bending sector; BS, bending sector; BBS, below bending sector; CX, convex side; CE, concave side.

Figure 7

Model summarizing correlation among anatomical, phytohormonal and lignin dataset in bent stem and root. The correlation among main anatomical parameters (CCN, RXT, and RPT – red vectors), phytohormones (IAA – green vectors; CKs free forms – cZ, cZR, tZ, tZR, iP, iPR, and DHZ – blue vectors) and total lignin content (Lignin – purple vectors) were analyzed by using Principal Component Analysis (PCA). Scatter plots show data variability within each sector (ABS, BS and BBS) of bent stem and root. Data were computed by using FactoMineR package in R and plotted by the two first principal components (PC1 and PC2). Vectors indicate direction and strength of each variable to the overall distribution. CCN, cambial cell number; CKs, cytokinins; cZ, cis-zeatin; cZR, cis-zeatin riboside; DHZ, dihydrozeatin; IAA, indole-3-acetic acid; iP, N-isopentenyladenine; iPR, N⁶-isopentenyladenosine; RXT, relative xylem thickness; RPT, relative phloem thickness; tZ, trans-zeatin; tZR, trans-zeatin riboside.